Project Summary The major cellular mRNA export factor Mex67/NXF1 (yeast/vertebrates) transports its cargo from the nucleus through the nuclear pore complex (NPC) to the cytoplasm in a distinct ATP dependent pathway, that is different from all other export factors that rely on the GTP/GDP-dependent Ran cycle. In most eukaryotes, Mex67/NXF1 exists as a single protein; however, additional tissue-specific isoforms of NXF1 exist in metazoa (i.e. humans). The functions of these tissue-specific isoforms have been difficult to delimit due to the complexity of control of gene expression in metazoa as well as the lack of simple model systems that may mimic different tissues. We recently discovered that Trypanosomatids, a group of divergent unicellular protozoans, have two very distinct paralogs of Mex67 with differing roles in mRNA export. Trypanosomatids lack individual gene promoter control, instead relying heavily on post-transcriptional gene regulation and potentially, placing RNA export at the center of the control of gene expression. Indeed, our preliminary results indicate that the two Mex67 paralogs have life cycle-specific roles, reminiscent of the tissue-specific NXF1 variants observed in metazoa. It is our hypothesis that these two Mex67 paralogs function to help differentially regulate the expression of genes in different life cycle stages. To determine this, we will quantitatively and comprehensively characterize the paralog-specific protein interactomes and RNA cargos of the two Mex67 proteins in cells in the different life stages by utilizing and adapting methods we have pioneered, as well as standard biochemical techniques. Concomitant with an expanded role of nuclear transport in gene regulation, we have previously shown that the trypanosomatid NPC lacks the entire ATP-dependent export machinery required to drive mRNA export in yeast and metazoa. Instead, our results suggest organisms depend on the Ran GTPase system, like all other transport pathways within the cell, a major departure from the canonical textbook model of mRNA export. We hypothesize that this system will provide a new perspective on how Ran can be utilized to mediate directional transport across the NPC. Our strategy will include classical biochemical techniques involving exogenously expressed components that have been succesfully employed to delimit nucleocytoplasmic transport in yeast and humans, as well as state of the art proteomic methods to compute topological maps of the TbMex67-Ran machinery. Our results will help reveal how different organisms and tissues may use export as an important mechanism to control gene expression, and how Ran may drive different nuclear transport pathways in previously unanticipated ways.